Dectin-1-Dependent Interleukin-22 Contributes to Early Innate Lung
Defense against Aspergillus fumigatus
Melissa A. Gessner,aJessica L. Werner,aLauren M. Lilly,aMichael P. Nelson,aAllison E. Metz,aChad W. Dunaway,aYvonne R. Chan,b
Wenjun Ouyang,cGordon D. Brown,dCasey T. Weaver,eand Chad Steelea
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USAa; Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania,
USAb; Department of Immunology, Genentech, Inc., South San Francisco, California, USAc; Section of Infection and Immunity, Institute of Medical Sciences, University of
Aberdeen, Aberdeen, United Kingdomd; and Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USAe
gillus fumigatus lung infection in the presence of lower interleukin 23 (IL-23) and IL-17A production in the lungs and have re-
Dectin-1 in IL-22 production, as well as the role of IL-22 in innate host defense against A. fumigatus. Here, we show that Dectin-
1-deficient mice demonstrated significantly reduced levels of IL-22 in the lungs early after A. fumigatus challenge. Culturing cells
from enzymatic lung digests ex vivo further demonstrated Dectin-1-dependent IL-22 production. IL-22 production was addi-
IL-22 production in wild-type (WT) lung cells and rescued IL-22 production by lung cells from Dectin-1-deficient mice. In vivo
neutralization of IL-22 in the lungs of WT mice resulted in impaired A. fumigatus lung clearance. Moreover, mice deficient in
IL-22 also demonstrated a higher lung fungal burden after A. fumigatus challenge in the presence of impaired IL-1?, tumor ne-
production. We further show that lung lavage fluid collected from both A. fumigatus-challenged Dectin-1-deficient and IL-22-
deficient mice had compromised anti-fungal activity against A. fumigatus in vitro. Although lipocalin 2 production was ob-
served to be Dectin-1 and IL-22 dependent, lipocalin 2-deficient mice did not demonstrate impaired A. fumigatus clearance.
Collectively, our results indicate that early innate lung defense against A. fumigatus is mediated by Dectin-1-dependent IL-22
life-threatening infections in patients who are severely immuno-
dysfunction, and immunosuppressive therapies (19). Recent data
from the Transplant Associated Infections Surveillance Network
(TRANSNET), a network of 23 United States transplant centers,
have shown that IA occurred in 43% of hematopoietic stem cell
transplant (HSCT) recipients (20) and in 19% of solid organ
transplant recipients (33) between March 2001 and March 2006.
IA is also becoming more recognized in individuals with less se-
vere levels of immunosuppression. This is increasingly observed
in intensive-care unit (ICU) populations, often associated with
cirrhosis, alcoholism, and postinfluenza infection; various post-
granulomatous disease (reviewed in reference 1).
Our laboratory has had a longstanding interest in pulmonary
We have previously demonstrated a central role for the beta-
glucan receptor Dectin-1 in innate lung immune responses
against A. fumigatus (38). Mice deficient in Dectin-1 are highly
susceptible to lung infection with A. fumigatus as a result of im-
paired inflammatory reactivity of alveolar macrophages and im-
paired recruitment of and defense by neutrophils (42). Among
several cytokines we have reported to be induced in a Dectin-1-
dependent manner during A. fumigatus lung infection, we have
identified interleukin 17A (IL-17A) as a critical mediator in host
spergillus fumigatus, the etiological agent of invasive aspergil-
losis (IA), is a ubiquitous mold that causes severe, invasive,
defense (42). Dectin-1-deficient mice produced IL-17A at lower
levels in the lungs after exposure to A. fumigatus, and neutraliza-
ability to clear A. fumigatus from the lungs, indicating a strong
requirement for the mediator in pulmonary defense against A.
fumigatus (42). In our most recent studies, we have identified
during A. fumigatus lung infection (43). IL-17A production by
neutrophils required the presence of IL-23, which we have previ-
the lungs (42) and, more recently, in a Dectin-1-dependent man-
ner by dendritic cells (DCs) (43).
In addition to being critical for the maintenance of the Th17 lin-
?-helical cytokine of the IL-10 family of cytokines (34). Although
often produced in concert with each other, growing data in several
Received 13 September 2011 Returned for modification 5 October 2011
Accepted 15 October 2011
Published ahead of print 28 October 2011
Editor: G. S. Deepe, Jr.
Address correspondence to Chad Steele, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
iai.asm.org 0019-9567/12/$12.00Infection and Immunityp. 410–417
function equally. For example, mucosal defense against the gut
pathogen Citrobacter rodentium is more dependent on IL-23 than
IL-17A (26). In colitis, IL-23 deficiency is more effective in amelio-
fection models with the Gram-negative bacteria Klebsiella pneu-
moniae (2) and C. rodentium (50) showed a requisite role for IL-22
in protection. However, although produced by Th17/IL-17A-
producing cells (29), IL-22 has been shown to act as an anti-
in inflammation associated with psoriasis (49). Collectively, IL-23,
in certain models, the function of one may be more important than
that of another. As we had previously identified a role for IL-17A in
MATERIALS AND METHODS
for 10 generations to the C57BL/6 background, and bred at Taconic. IL-22-
at the University of Alabama at Birmingham (UAB). Lipocalin 2-deficient
were maintained in a specific-pathogen-free environment in microisolator
cages within an American Association for Laboratory Animal Science-
certified animal facility in the Lyons Harrison Research Building at the Uni-
versity of Alabama at Birmingham. Animal studies were reviewed and ap-
proved by the University of Alabama at Birmingham Institutional Animal
den assessment. A. fumigatus isolate 13073 (ATCC, Manassas, VA) was
maintained on potato dextrose agar for 5 to 7 days at 37°C. Conidia were
harvested by washing the culture flask with 50 ml of sterile phosphate-
passed through a sterile 40-?m nylon membrane to remove hyphal frag-
ments and enumerated on a hemacytometer. Mice were lightly anesthe-
tized with isoflurane and administered 5 ? 107to 7 ? 107A. fumigatus
ysis, the left lungs were collected and homogenized in 1 ml of phosphate-
lung homogenate using the MasterPure yeast RNA purification kit (Epi-
step to eliminate genomic DNA, as previously reported (28). The lung A.
fumigatus 18S rRNA (GenBank accession number AB008401) (5) and
quantified using a standard curve for A. fumigatus conidia (101to 109) as
previously described (28). Specifically, total RNA was isolated, using the
MasterPure kit, from serial 1:10 dilutions of A. fumigatus conidia, begin-
was performed on each dilution. As a validation of the real-time PCR
method, heat-killed A. fumigatus did not yield a signal by real-time PCR
and was unable to grow on potato dextrose agar plates. In addition, no
reaction mixture) yielded a signal of ?0.001% by real-time PCR, indicat-
ing that the DNase treatment step was efficient at eliminating contami-
nating A. fumigatus DNA.
IL-22 neutralization. For in vivo IL-22 neutralization, WT mice were
50 ?l, and 6 h thereafter, the mice were administered 50 ?g of goat anti-
mouse IL-22 (R&D Systems) or goat IgG isotype control antibody.
Twenty-four hours after challenge, the mice were sacrificed, and the left
lungs were collected and homogenized in 1 ml of PBS. Total RNA was
extracted from 0.1 ml of unclarified lung homogenate using the Master-
Pure yeast RNA purification kit, and the lung A. fumigatus burden was
as described previously (42).
Lung cell isolation, culture, cytokine neutralizations, and IL-23
stimulation. Mice were anesthetized with intraperitoneal ketamine/xyla-
collected and minced in Iscove’s modified Dulbecco’s medium (IMDM)
(Sigma, St. Louis, MO) supplemented with 1% penicillin-streptomycin-
Fisher), followed by incubation for 60 min with tissue culture grade type IV
collagenase (1 mg/ml; Sigma, St. Louis, MO) in a 37°C orbital shaker at 100
lon filters, and red blood cells were lysed with ACK buffer (Lonza, Walkers-
ville, MD) to create lung cell preparations. For lung cell cultures, cells were
bent assay (ELISA) (42). In specific experiments, neutralizing antibodies
were added to lung cells to assess the effects of cytokine neutralization on
IL-22 production. For this, anti-mouse IL-1?, IL-6, IL-18, and IL-23 (all
from R&D Systems) were added to lung cell cultures at a final concentra-
tion of 2 to 5 ?g/ml for 24 h. Rat (IL-1?, IL-6, and IL-18) or goat (IL-23)
isotype antibodies were added to lung cell cultures as a control. The su-
pernatants were collected after 24 h and clarified by centrifugation, and
IL-22 levels were quantified by ELISA (R&D Systems). In specific experi-
ments, recombinant murine IL-23 (R&D Systems) was added to lung
24 h and clarified by centrifugation, and IL-22 levels were quantified by
ELISA (R&D Systems). For lung neutrophil analysis by flow cytometry,
(BD Biosciences, San Diego, CA) at 4°C for 20 min. Thereafter, the cells
vitrogen), followed by labeling with CD11b?and Ly6G?(1A8 clone;
antibodies from BD Biosciences) (43).
Analysis of lung lavage fluid antifungal activity. Wild-type, Dectin-
a bronchoalveolar lavage was performed as previously described (31, 38).
fumigatus. Fifty microliters of clarified lavage fluid from each strain was
incubated with 1 ? 105A. fumigatus conidia (in 150 ?l of RPMI supple-
mented with 10% FBS and 1% penicillin-streptomycin) for 4 h at 37°C.
Thereafter, the contents of the well was subjected to total RNA extraction
using the MasterPure yeast RNA purification kit and analyzed for A. fu-
migatus viability as described above. RNA was also extracted from the
lavage fluid to assess the presence of A. fumigatus after centrifugation,
which demonstrated negligible levels (4 to 5 log units below that quanti-
fied in 50 ?l lavage fluid plus 1 ? 105A. fumigatus conidia).
Lipocalin 2, S100a8, S100a9, and Reg3g analysis. C57BL/6 WT,
were collected and homogenized in TRIzol reagent (Invitrogen), and total
RNA was isolated according to the manufacturer’s instructions. Thereafter,
RNA was transcribed to cDNA (iScript cDNA synthesis kit; Bio-Rad), and
real-time PCR for S100a8 (Mm00496696_g1; Applied Biosystems), S100a9
(Mm00656925_m1; Applied Biosystems), and Reg3g (Mm00441127_m1;
Applied Biosystems) was performed (iQ Supermix; Bio-Rad). mRNA levels
were normalized to Gapdh mRNA levels (primers/probe from Applied Bio-
systems) using the 2???CTmethod. For lipocalin 2 quantification, C57BL/6
WT, Dectin-1-deficient, and IL-22-deficient mice were challenged intratra-
Innate Lung Immunity to A. fumigatus
January 2012 Volume 80 Number 1 iai.asm.org 411
the left lungs were homogenized in PBS supplemented with Complete Mini
protease inhibitor tablets (Roche), clarified by centrifugation, and stored at
Statistics. Data were analyzed using GraphPad Prism Version 5.0 sta-
tistical software. Comparisons between groups when data were normally
P value of ?0.05.
IL-22 production after A. fumigatus challenge is dependent
on Dectin-1. As we have previously reported that Dectin-1-
dependent IL-17A was a critical component of lung defense
against A. fumigatus (42), we sought to determine whether IL-22
was also dependent on beta-glucan recognition via Dectin-1 and
whether it was required for A. fumigatus host defense. The results
in Fig. 1A show that IL-22 was robustly induced in the lungs after
not shown]) and required Dectin-1-mediated recognition of A.
fumigatus, as mice deficient in Dectin-1 had severely compro-
mised production of IL-22 in the lungs. We next collected lungs
from C57BL/6 (WT) and Dectin-1-deficient (knockout [KO])
mice 18 h after A. fumigatus challenge and subjected them to en-
zymatic digestion to determine whether single-cell suspensions
could replicate the differences in IL-22 levels observed in whole-
lung homogenates, as we have recently reported for IL-17A (43).
Upon ex vivo culturing of lung cells overnight, cells from Dectin-
1-deficient mice had a ?8-fold reduction in IL-22 production
compared to WT lung digest cells (Fig. 1B). Thus, beta-glucan
recognition via Dectin-1 mediates lung IL-22 production after A.
IL-22 is required for early A. fumigatus lung clearance. IL-
17A is acknowledged to stimulate the antimicrobial immune ef-
fector functions of multiple cell types, including neutrophils,
macrophages, and epithelial cells (12). We have previously re-
of A. fumigatus (42). However, as IL-22 appears to primarily acti-
of IL-22, based on its limited cellular targeting, would have a sig-
nificant effect on innate immune clearance of A. fumigatus. The
results in Fig. 2 show that neutralization of IL-22 in the lungs of
C57BL/6 mice (Fig. 2A) resulted in a ?5-fold increase in the A.
fumigatus lung burden by 24 h postinfection (Fig. 2B). We con-
fection (Fig. 3A). Despite having higher A. fumigatus lung bur-
of multiple cytokines and chemokines previously implicated in
FIG 1 IL-22 production after A. fumigatus challenge is dependent on
intratracheally with 5 ? 107to 7 ? 107A. fumigatus conidia, and 48 h after
exposure, IL-22 levels were quantified in lung homogenates by ELISA. The
data are expressed as mean pg/ml plus standard error of the mean (SEM).
for each study). ??? indicates a P value of ?0.001 (unpaired two-tailed Stu-
dent’s t test). (B) C57BL/6 WT and Dectin-1-deficient (KO) mice were chal-
lenged intratracheally with 5 ? 107to 7 ? 107A. fumigatus conidia, and 18 h
after exposure, lungs were collected and enzymatically digested. Single-cell
of 0.2 ml. IL-22 levels were quantified in clarified coculture supernatants by
ELISA. Shown are cumulative data from four independent studies. The data
are expressed as mean pg/ml plus SEM. ??? indicates a P value of ?0.001
(unpaired two-tailed Student’s t test).
of ?0.01 (unpaired two-tailed Student’s t test). (B) Real-time PCR analysis of
or isotype control antibodies. Shown are cumulative data from two indepen-
dent studies (n ? 5 mice per group per time point). The data are expressed as
mean A. fumigatus 18S rRNA plus SEM. ?? indicates a P value of ?0.01 (un-
paired two-tailed Student’s t test).
Gessner et al.
iai.asm.orgInfection and Immunity
lung host defense against A. fumigatus, including IL-1?, tumor
necrosis factor alpha (TNF-?), CCL3/MIP-1?, and CCL/
4MIP-1? (13, 30) (Fig. 3B). In turn, the lack of these proinflam-
in CD11b?Ly-6G?neutrophils in the lungs of IL-22-deficient
mice (Fig. 3C). We also observed reductions in IL-12p40 and IL-
12p70 (Fig. 3D), although gamma interferon (IFN-?) levels were
CXCL9/Mig production in the lungs during bacterial pneumonia
to have direct antimicrobial activity (9), suggesting the possibility
as an innate effector molecule against A. fumigatus. However, IL-
22-deficient mice exposed to A. fumigatus did not demonstrate a
reduction in CXCL9/Mig or CXCL10/IP-10 (Fig. 3E), diminish-
ing the likelihood of a role for these molecules in IL-22-mediated
defense against A. fumigatus. Intriguingly, IL-17A levels were sig-
deficient mice (Fig. 3F), indicating that IL-17A production in the
lungs is not dependent on IL-22. Thus, IL-22 is required for opti-
mal clearance of A. fumigatus from the lungs.
Impaired antifungal activity in lung lavage fluid from A.
fumigatus-challenged Dectin-1-deficient and IL-22-deficient
mice. Mice deficient in the beta-glucan receptor Dectin-1 have
FIG 3 IL-22-deficient mice have impaired A. fumigatus lung clearance. (A) C57BL/6 WT and IL-22-deficient (IL-22 KO) mice were challenged intratracheally
with 5 ? 107to 7 ? 107A. fumigatus conidia, and 24 h after exposure, the lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA
indicates a P value of ?0.01 (unpaired two-tailed Student’s t test). (B) Levels of IL-1?, TNF-?, CCL3, and CCL4 were quantified in lung homogenates collected
pg/ml plus SEM. ? and ??? indicate P values of ?0.05 and ?0.001, respectively (unpaired two-tailed Student’s t test). (C) Lung cells were isolated via
bronchoalveolar lavage, Fc blocked, stained with a LIVE/DEAD staining kit, and then stained with fluorochrome-conjugated CD11b and Ly-6G. Shown are
representative data from one of two independent studies. The data are expressed as the absolute number of live cells in lung lavage fluid. ? indicates a P value of
?0.05 (unpaired two-tailed Student’s t test). (D) IL-12p40 and IL-12p70 were quantified in lung homogenates collected 24 h postinfection by Bio-Plex. Shown
are cumulative data from three independent studies (n ? 5 mice per group per time point). The data are expressed as mean pg/ml plus SEM. ? and ?? indicate
are expressed as mean pg/ml plus SEM. ?? indicates a P value of ?0.01 (unpaired two-tailed Student’s t test).
Innate Lung Immunity to A. fumigatus
January 2012 Volume 80 Number 1 iai.asm.org 413
response to A. fumigatus, and neutralization of IL-17A (42) or
IL-22 (Fig. 2) compromises clearance of A. fumigatus from the
lungs. As both IL-17A and IL-22 are efficient at eliciting soluble
antimicrobial factors from epithelial cells (50), we hypothesized
that defects in these factors would be reflected in the antifungal
activity of lung lavage fluid. The results in Fig. 4A show that lung
lavage fluid from Dectin-1-deficient mice demonstrated poor an-
tifungal activity compared to lung lavage fluid from WT mice.
mised antifungal activity, although it was not at the level of
and an IL-22-dependent (Fig. 4B) induction of the siderophore
binding protein lipocalin 2, which can be induced by IL-17A and
own siderophores (35), we hypothesized that lipocalin 2 may act
as an antifungal agent against A. fumigatus by limiting A. fumiga-
tus iron acquisition. To our surprise, mice deficient in lipocalin 2
IL-22 has also been shown to induce other antimicrobial proteins
low induction in response to A. fumigatus (1.5- to 2-fold) but
intact expression in Dectin-1-deficient and IL-22-deficient mice
(data not shown). Similarly, S100a8 and S100a9 mRNA expres-
sion was induced 15- to 25-fold in response to A. fumigatus but
was not modulated in Dectin-1-deficient or IL-22-deficient mice
(data not shown). Thus, one mechanism of susceptibility to A.
fumigatus in the setting of Dectin-1 or IL-22 deficiency is a puta-
tive lack of or impairment in a soluble factor(s) with antifungal
activity; however, this factor(s) does not appear to be lipocalin 2,
S100A8, S100A9, or RegIII?.
independent of IL-1?, IL-6, and IL-18 but requires IL-23. We
have previously employed the culture system in Fig. 1B to deter-
mine mechanisms associated with Dectin-1-dependent IL-17A
production (43). IL-22 is recognized to be produced by IL-17A-
producing CD4 T cells (Th17 cells), although other cellular
sources have been described (27, 40). Along with IL-17A produc-
tion, cytokines, such as IL-6, IL-23, and IL-1?, have also been
(21). In addition, IL-18 may synergize with IL-12 or IL-23 for
neutralization of IL-23 in lung cell cultures from A. fumigatus-
challenged mice resulted in attenuated IL-17A production (43).
Moreover, IL-23p19-deficient mice have reduced IL-22 produc-
fore, we questioned whether lung IL-22 production was similarly
dependent on IL-1?, IL-6, IL-18, or IL-23 during A. fumigatus
infection. We have previously reported that IL-6 and IL-1? were
produced at lower levels by lung cells from Dectin-1-deficient
ever, neutralization of IL-1?, IL-6, or IL-18 did not significantly
reduce lung cell production of IL-22 (Fig. 5A). Although IL-1?
neutralization appeared to lower IL-22 production, it did not
reach statistical significance (P ? 0.0685). Once again, however,
IL-23 was a key factor in IL-22 induction, as neutralization of
lung cell cultures resulted in increased IL-22 production, even in
FIG 4 Impaired antifungal activity in lung lavage fluid from A. fumigatus-
challenged Dectin-1-deficient and IL-22-deficient mice. (A) C57BL/6 WT,
Dectin-1-deficient (Dectin-1 KO), and IL-22-deficient (IL-22 KO) mice
were challenged intratracheally with 5 ? 107to 7 ? 107A. fumigatus
conidia, and 24 h after exposure, bronchoalveolar lavage was performed.
The lung lavage fluid was processed to remove cells and A. fumigatus, and
then, 50 ?l of clarified lavage fluid from each strain was incubated with 1 ?
105A. fumigatus conidia (in 150 ?l of RPMI supplemented with 10% FBS
the well was subjected to total RNA extraction using the MasterPure yeast
RNA purification kit and analyzed for A. fumigatus viability. For each
experiment, the percent above WT was calculated by dividing the A. fu-
migatus 18S rRNA units in Dectin-1-deficient and IL-22-deficient cultures
by the A. fumigatus 18S rRNA units in WT cultures. WT values were set at
100. Shown are cumulative data from eight independent studies. ? and ???
indicate P values of ?0.05 and ?0.001, respectively (paired two-tailed
Student’s t test). (B) C57BL/6 WT, Dectin-1-deficient, and IL-22-deficient
mice were challenged intratracheally with 5 ? 107to 7 ? 107A. fumigatus
conidia, and 24 h after exposure, lungs were collected and homogenized
and lipocalin 2 levels were quantified in the clarified homogenates by
ELISA. Shown are cumulative data from two independent studies (n ? 4 to
5 per group). ?? and ??? indicate P values of ?0.01 and ?0.001, respec-
2-deficient (Lcn2 KO) mice were challenged intratracheally with 5 ? 107to
7 ? 107A. fumigatus conidia, and 24 h after exposure, the lung fungal
burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA
levels. Shown are cumulative data from two independent studies (n ? 5
mice per group). The data are expressed as mean A. fumigatus 18S rRNA
Gessner et al.
iai.asm.org Infection and Immunity
lung cells from Dectin-1-deficient mice. Thus, IL-22 production
dent on IL-23, and IL-23 can restore IL-22 production in Dectin-
studies have discovered that CD4?T cells producing IL-17A can
also produce the cytokine IL-22, a member of the IL-10 family of
cytokines (24). Analogous to the observations for IL-17A, addi-
tional cell types, such as lymphoid tissue inducer cells (40), NK
cells (6), and ?? T cells (27), can also produce IL-22. Although
in stimulating epithelial antimicrobial activity and host defense
against multiple mucosal pathogens (50), including the fungal
organism Candida albicans (10). To date, only epithelial cells and
IL-17A in host defense against A. fumigatus (42, 43). As IL-17A
required for IL-22 induction in several models (10, 37, 40), we
induction of IL-22 and the role of IL-22 in A. fumigatus lung
fumigatus challenge. In both lung homogenates and lung cell cul-
tures from Dectin-1-deficient mice, IL-22 was produced at less
that some of this is due to compromised IL-23 production in
Dectin-1-deficient mice (43), the dependency of IL-22 on
Dectin-1 during A. fumigatus exposure is more striking than that
observed in IL-23-deficient mice systemically exposed to C. albi-
levels (10). Coupling this observation with our data indicating
that IL-22 production by lung cells is reduced by three-fourths in
sites of infection, additional mediators are likely involved in opti-
mal IL-22 production (i.e., the remaining quarter to a third in
cytokines had no effect on IL-22 production by lung cells from A.
fumigatus-exposed mice. As IL-23 signals through IL-12R?1 and
IL-23R?, it is thought to activate the STAT1, STAT4, STAT3, and
STAT5 signaling pathways (14). With respect to Th17/IL-17A re-
STAT3, and possibly other STATs as well, and synergize with
IL-23 for optimal lung IL-22 production. Currently, studies are
IL-22 production by lung cells.
To thoroughly examine the role of IL-22 in lung host defense
designs: (i) neutralization and (ii) genetic deficiency. Neutraliza-
tion of IL-22, as well as IL-22 deficiency, led to significantly com-
promised clearance of A. fumigatus from the lungs. The level of
impairment in fungal clearance was also more apparent with A.
fumigatus than in a previous report with C. albicans, which dem-
onstrated 2-fold changes in stomach (gastrointestinal infection)
and kidney (systemic infection) burdens (3 days postchallenge)
when IL-22 was genetically deficient (10). Neutralization of IL-22
in this model had little or no effect on the C. albicans stomach
kidney burden by only a third in BALB/c mice (10). In contrast,
our studies revealed that IL-22 neutralization resulted in a 5-fold
increase in the lung A. fumigatus burden, whereas IL-22 genetic
deficiency resulted in an 8-fold increase in the A. fumigatus bur-
den. There are many possibilities as to why differences were ob-
pathogens are quite different in their tissue specificities and host
defense requirements; thus, it is possible that host defense against
one organism may require IL-22 more than the other. Moreover,
our studies investigated the role of IL-22 in early/rapid host de-
fense against A. fumigatus, i.e., 1 to 2 days postchallenge, in con-
FIG 5 IL-22 production by lung cells in response to A. fumigatus is indepen-
dent of IL-1?, IL-6, and IL-18 but requires IL-23. (A) Lung cells were isolated
as described in Materials and Methods, and 1 ? 106cells were cultured for 24
culture. Rat (IL-1?, IL-6, and IL-18) or goat (IL-23) isotype antibodies were
natants by ELISA. Shown are cumulative data from two independent studies
for each condition (isotype and neutralizing antibody) run in triplicate. The
(unpaired two-tailed Student’s t test); ns, not significant. (B) Lung cells were
isolated from WT and KO mice as described, and 1 ? 106cells were cultured
for 24 h in a volume of 0.2 ml. Recombinant murine IL-23 was added at 1 and
10 ng/ml at the beginning of the culture. The controls included lung cells
cultured in the absence of IL-23. IL-22 levels were quantified in clarified co-
culture supernatants by ELISA. Shown are cumulative data from three inde-
pendent studies. The data are expressed as mean pg/ml plus SEM. ?, ??, and
Student’s t test).
Innate Lung Immunity to A. fumigatus
January 2012 Volume 80 Number 1 iai.asm.org 415
addition, it is also possible that the role of IL-22 may be more
evident, perhaps even more important, in such tissues as the lung
and gut, where the overwhelming majority of cells are epithelial
cells and keratinocytes. Nevertheless, our studies point to an es-
sential role for IL-22 at the earliest stages of A. fumigatus lung
As mentioned previously, we have documented a role for IL-
17A in A. fumigatus lung defense (42) and now extend this to
lung infection with K. pneumoniae (17, 2) and gut infection with
not always play equal roles in host defense. Cutaneous infection
is correlated with a lack of IL-17A, but not IL-22, production (7).
In models of oral infection (8) and skin infection (18) with C.
albicans, IL-17A, but not IL-22, was required for defense. Protec-
tive immunity to systemic infection with attenuated Salmonella
enterica serovar Enteritidis is associated with IL-22, but not IL-17
(36), while infection with Borrelia burgdorferi induces a potent
IL-22 response, yet IL-17A is completely absent (3). Finally, in
Listeria monocytogenes infection, IL-17A is required for clearance
(16), but not IL-22 (15), a finding also observed in Francisella
tularensis infection (25). However, during A. fumigatus lung in-
increased in the lungs of IL-22-deficient mice challenged with A.
fumigatus, yet lung clearance is impaired. In turn, we have re-
ported that IL-17A neutralization leads to impaired A. fumigatus
lung clearance (42), although IL-22 levels were not affected by
98 pg/ml, n ? 10, in lung homogenates for isotype- and anti-IL-
17A-treated mice, respectively). Therefore, in a scenario where
ing response is not sufficient to compensate.
is in the induction of the epithelial antimicrobial response. Initial
studies examining the function of IL-22 showed that stimulation
teins, and RegIII proteins (50). IL-17A also has an acknowledged
role in the induction of these factors (2), and IL-22 can often add
to or synergize with IL-17A for the induction of the epithelial
antimicrobial response. Recognizing that IL-22, along with IL-
17A, can evoke this response in the lungs (2) led us to determine
whether functional defects existed in the lungs of Dectin-1-
end, we demonstrated that clarified lung lavage fluid (i.e., fluid
that was free of live A. fumigatus and live host cells) from both
Dectin-1-deficient and IL-22-deficient mice did not kill A. fu-
1-deficient mice, which we hypothesize is a result of these mice
having significant reductions in both IL-17A (42) and IL-22 (Fig.
1). Despite compromised S100A8 and S100A9 expression in IL-
lungs of A. fumigatus-exposed Dectin-1-deficient and IL-22-
found to be statistically lower in the lungs (data not shown). In
contrast, we did observe a reduction in the lung levels of lipocalin
2, a siderophore binding protein induced by IL-22 (2), in both
lin 2-deficient mice did not demonstrate an impairment in A.
fumigatus lung clearance, indicating that lipocalin 2 does not ap-
an effect of lipocalin 2 deficiency on A. fumigatus lung clearance,
we cannot exclude the possibility that other antimicrobial factors
are compensating for the loss of lipocalin 2. For example, lacto-
ferrin can mediate reactive oxygen species (ROS)-independent
killing of A. fumigatus by neutrophils (46). Currently, studies are
under way to identify the Dectin-1- and IL-22-dependent soluble
antifungal factors induced in the lungs during A. fumigatus infec-
In summary, we have identified a role for IL-22 in early innate
beta-glucan receptor Dectin-1. Both neutralization of and genetic
tory cytokines and chemokines, as well as the lung antifungal
response. However, the Dectin-1- and IL-22-dependent lung anti-
fungal response was independent of the known IL-17A- and IL-22-
associated antimicrobial factor S100 proteins, RegIII? and lipocalin
dependent antifungal mechanism. As with our recent report on IL-
to IL-23, may also play a role in Dectin-1-dependent IL-22 produc-
taneously needed for A. fumigatus lung clearance and that IL-23 is
infection, IL-23 may be an effective immunotherapy for the treat-
the IL-23/IL-17A/IL-22 axis in innate lung defense against A. fumi-
This work was supported was supported by Public Health Service grants
AI068917 and HL096702.
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Innate Lung Immunity to A. fumigatus
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